Transforming Growth Factor-beta (TGF-β) is a powerful signaling protein, a cytokine, that influences a wide array of biological processes within the body. Its multifaceted roles include regulating how cells grow, specialize, and interact with their surroundings. Researchers frequently utilize recombinant forms of this protein to study these complex cellular mechanisms in a controlled laboratory setting. PeproTech is a recognized manufacturer that provides such recombinant proteins, including various TGF-β isoforms, for scientific investigations.
The Biological Role of the TGF-β Superfamily
The TGF-β superfamily encompasses a group of related proteins that govern fundamental cellular behaviors in the body. TGF-β is a polypeptide cytokine found in tissues. It is involved in regulating cell proliferation, differentiation, adhesion, and migration.
TGF-β also plays a role in immune responses, modulating inflammation and immune cell development, including T and B lymphocytes. For example, it can inhibit B cell proliferation by influencing specific transcription factors and cell cycle regulators. Beyond immunity, TGF-β contributes to wound healing and embryonic development.
There are three main mammalian isoforms: TGF-β1, TGF-β2, and TGF-β3, which can exist as homodimers or heterodimers. These proteins are secreted in a latent form, complexed with other peptides, and require activation by proteases or cell surface receptors like integrins. Once active, TGF-β initiates signaling by binding to specific type I and type II serine/threonine kinase receptors on the cell surface. This binding leads to the activation of an intracellular signaling cascade, primarily through the canonical Smad pathway.
In the Smad pathway, activated type I receptors phosphorylate receptor-regulated Smads (R-Smads), such as Smad2 and Smad3. These phosphorylated R-Smads then associate with a common Smad (Co-Smad), Smad4, forming a complex that moves into the cell nucleus. Inside the nucleus, this Smad complex interacts with various transcription factors to regulate the expression of target genes, thereby orchestrating the diverse cellular responses attributed to TGF-β.
Product Specifications and Formulation
PeproTech’s recombinant human TGF-β1 is produced from HEK293 cells. This protein is a homodimer, with each subunit consisting of 112 amino acid residues and linked by a single disulfide bond, resulting in a molecular weight of approximately 25.0 kDa.
PeproTech’s TGF-β products are at least 98% pure, verified by SDS-PAGE and HPLC. Biological activity is determined through cell-based assays, such as the ability of TGF-β1 to inhibit the interleukin-4 (IL-4)-dependent proliferation of mouse HT-2 cells. The effective dose 50 (ED50) for this inhibition is up to 0.05 ng/ml, corresponding to a specific activity of at least 2 x 10^7 units/mg.
The product is supplied lyophilized (freeze-dried) from a buffered solution like sodium citrate at pH 3.5. This formulation helps maintain protein stability during shipment and storage. PeproTech also offers human and murine TGF-β versions. For instance, human and mouse TGF-β3 show 100% sequence homology and cross-species activity.
Handling and Experimental Application
Proper handling of PeproTech’s lyophilized TGF-β is important for maintaining its activity and stability. Before reconstitution, vials should be briefly centrifuged, around 13,000 rpm for 30-60 seconds, to ensure all the powder is at the bottom of the tube. Initial reconstitution should follow the lot-specific Certificate of Analysis (CoA), recommending a sterile buffer like 10 mM citric acid at pH 3.0, to achieve a stock concentration of 0.1-1.0 mg/ml. Avoid adding a carrier protein to this initial reconstitution unless stated on the CoA.
Once reconstituted, the protein should be mixed carefully by gently pipetting up and down or by inverting the vial a few times, avoiding vigorous vortexing. The reconstituted stock solution can be stored at 2-8°C for up to one week. For long-term storage, it is recommended to dilute the stock solution into a buffer or medium containing a carrier protein, such as 0.1% Bovine Serum Albumin (BSA) in Phosphate-Buffered Saline (PBS).
To prevent degradation and loss of activity, the diluted solution should be aliquoted into smaller volumes and stored at -20°C to -80°C for 3-12 months, as indicated on the CoA. Avoid repeated freeze-thaw cycles for protein integrity. Individual aliquots should have a concentration of at least 1 µg/mL and a volume of at least 10 μL. In cell culture applications, working concentrations of TGF-β vary by cell type and desired effect, ranging from picograms to nanograms per milliliter.
Quality Control and Validation
PeproTech implements thorough quality control measures to ensure the consistency and biological activity of its recombinant TGF-β products. Each lot undergoes rigorous testing to meet defined specifications. A primary measure involves verifying protein concentration and purity through SDS-PAGE and HPLC analyses.
Endotoxin levels are also determined, kept very low at less than 0.1 ng per microgram (1 EU/µg) of TGF-β1. Endotoxins can interfere with cell-based assays and induce unwanted cellular responses, so their low presence ensures experimental reliability. Bioassays are also performed to confirm the biological activity of each lot, such as the HT-2 cell proliferation inhibition assay for TGF-β1, which determines the ED50 and specific activity.
These validation processes collectively ensure that the recombinant TGF-β supplied by PeproTech is suitable for research applications. The detailed specifications and testing results for each product lot are provided in a Certificate of Analysis, allowing researchers to verify the quality and performance of their specific product. This commitment to quality control helps researchers achieve reproducible and accurate results in their studies.